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• W2053287601 abstract "The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy, nanoparticle-mediated miR-21 silencing constitutes a promising multimodal therapeutic approach towards GBM. The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy, nanoparticle-mediated miR-21 silencing constitutes a promising multimodal therapeutic approach towards GBM. IntroductionGlioblastoma (GBM) is the most common and lethal primary brain tumor in humans.1Ohgaki H Kleihues P Genetic pathways to primary and secondary glioblastoma.Am J Pathol. 2007; 170: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar Despite the ongoing research efforts, current treatment options for GBM are largely unsatisfactory and the prognosis is usually poor with a 9- to 12-month median survival time (following diagnosis) that has not improved significantly over the last decade.2Khasraw M Lassman AB Advances in the treatment of malignant gliomas.Curr Oncol Rep. 2010; 12: 26-33Crossref PubMed Scopus (91) Google Scholar,3Stupp R Hegi ME Mason WP van den Bent MJ Taphoorn MJ Janzer RC European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups; National Cancer Institute of Canada Clinical Trials Group et al.Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.Lancet Oncol. 2009; 10: 459-466Abstract Full Text Full Text PDF PubMed Scopus (5357) Google Scholar It is therefore important to develop new therapeutical strategies that could encompass both high specificity for tumor cells and complete tumor eradication.Driven by the tremendous advances in molecular biology, gene therapy constitutes an attractive approach for modulation of the cell genetic background. Regulators of gene transcription and translation operate at multiple levels in order to fine-tune the genome end products. MicroRNAs (miRNAs) are elements of this complex modulatory network that play a pivotal role in cell fate. Dysregulation of several miRNAs (such as miR-21) has indeed been associated with development and progression of several cancers, including GBM.4Novakova J Slaby O Vyzula R Michalek J MicroRNA involvement in glioblastoma pathogenesis.Biochem Biophys Res Commun. 2009; 386: 1-5Crossref PubMed Scopus (118) Google Scholar,5Volinia S Calin GA Liu CG Ambs S Cimmino A Petrocca F et al.A microRNA expression signature of human solid tumors defines cancer gene targets.Proc Natl Acad Sci USA. 2006; 103: 2257-2261Crossref PubMed Scopus (4893) Google Scholar Therefore, these small post-transcriptional regulators constitute novel and highly promising targets for antitumoral strategies.Due to their unique characteristics (low size, low immunogenicity, high target affinity), antisense oligonucleotides (asOs) constitute an important tool for the manipulation of miRNA function in biological systems. In this regard, recent studies (including those reported in our previous manuscript) have shown that miRNA modulation in GBM cells results in decreased tumor cell migration and proliferation, as well as increased cytotoxic effect of antineoplastic drugs.6Costa PM Cardoso AL Nóbrega C Pereira de Almeida LF Bruce JN Canoll P et al.MicroRNA-21 silencing enhances the cytotoxic effect of the antiangiogenic drug sunitinib in glioblastoma.Hum Mol Genet. 2013; 22: 904-918Crossref PubMed Scopus (67) Google Scholar,7Dong CG Wu WK Feng SY Wang XJ Shao JF Qiao J Co-inhibition of microRNA-10b and microRNA-21 exerts synergistic inhibition on the proliferation and invasion of human glioma cells.Int J Oncol. 2012; 41: 1005-1012Crossref PubMed Scopus (72) Google Scholar,8Qian X Ren Y Shi Z Long L Pu P Sheng J et al.Sequence-dependent synergistic inhibition of human glioma cell lines by combined temozolomide and miR-21 inhibitor gene therapy.Mol Pharm. 2012; 9: 2636-2645Crossref PubMed Scopus (63) Google Scholar Nevertheless, the successful therapeutic application of oligonucleotide-based therapies to brain cancer requires novel strategies to overcome the barriers imposed by this complex organ. The presence of the blood–brain barrier, which restricts entry of therapeutic molecules into the brain,9Catuogno S Esposito CL Quintavalle C Condorelli G de Franciscis V Cerchia L Nucleic acids in human glioma treatment: innovative approaches and recent results.J Signal Transduct. 2012; 2012: 735135Crossref PubMed Google Scholar and the possible degradation of nucleic acids by nucleases present in the blood constitute major obstacles associated with nucleic acid delivery in vivo. It is, therefore, crucial that oligonucleotides are properly delivered by vehicles that are not only reliable and effective in overcoming cellular and physiological barriers, but are also highly target specific. Carrier systems, such as viruses or liposomes, have been developed to ensure protection and improvement of nucleic acid delivery into target cells.10Cardoso AL Simões S de Almeida LP Plesnila N Pedroso de Lima MC Wagner E et al.Tf-lipoplexes for neuronal siRNA delivery: a promising system to mediate gene silencing in the CNS.J Control Release. 2008; 132: 113-123Crossref PubMed Scopus (70) Google Scholar,11Hutterer M Gunsilius E Stockhammer G Molecular therapies for malignant glioma.Wien Med Wochenschr. 2006; 156: 351-363Crossref PubMed Scopus (19) Google Scholar,12Kanai R Rabkin SD Yip S Sgubin D Zaupa CM Hirose Y et al.Oncolytic virus-mediated manipulation of DNA damage responses: synergy with chemotherapy in killing glioblastoma stem cells.J Natl Cancer Inst. 2012; 104: 42-55Crossref PubMed Scopus (80) Google Scholar Recently, a new class of nucleic acid lipid particles, designated stable nucleic acid lipid particles (SNALPs), were shown (by us and other groups) to be very efficient in delivering small interfering RNAs (siRNAs), both in vitro and in vivo.13Gomes-da-Silva LC Santos AO Bimbo LM Moura V Ramalho JS Pedroso de Lima MC et al.Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment.Int J Pharm. 2012; 434: 9-19Crossref PubMed Scopus (39) Google Scholar,14Judge AD Robbins M Tavakoli I Levi J Hu L Fronda A et al.Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.J Clin Invest. 2009; 119: 661-673Crossref PubMed Scopus (286) Google Scholar,15Mendonça LS Firmino F Moreira JN Pedroso de Lima MC Simões S Transferrin receptor-targeted liposomes encapsulating anti-BCR-ABL siRNA or asODN for chronic myeloid leukemia treatment.Bioconjug Chem. 2010; 21: 157-168Crossref PubMed Scopus (79) Google Scholar Targeted therapy using peptides coupled to liposomal systems towards overexpressed tumoral receptors enables tumor-specific delivery, while minimizing side effects to normal cells. In this regard, chlorotoxin (CTX), a scorpion-derived peptide, was reported as a specific marker for gliomas16Mamelak AN Jacoby DB Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin (TM-601).Expert Opin Drug Deliv. 2007; 4: 175-186Crossref PubMed Scopus (209) Google Scholar and is currently used as a targeting agent in imaging studies (as well as in delivery of RNA interference therapeutics).17Veiseh O Kievit FM Fang C Mu N Jana S Leung MC et al.Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery.Biomaterials. 2010; 31: 8032-8042Crossref PubMed Scopus (159) Google Scholar,18Veiseh O Sun C Fang C Bhattarai N Gunn J Kievit F et al.Specific targeting of brain tumors with an optical/magnetic resonance imaging nanoprobe across the blood-brain barrier.Cancer Res. 2009; 69: 6200-6207Crossref PubMed Scopus (317) Google Scholar Moreover, CTX was reported to bind to matrix metalloproteinase-2 that is specifically upregulated in gliomas and related cancers, but poorly expressed in brain and normal tissues.19Deshane J Garner CC Sontheimer H Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2.J Biol Chem. 2003; 278: 4135-4144Crossref PubMed Scopus (332) Google ScholarIn this work, we employed CTX as a ligand to design targeted SNALPs for the delivery of asOs and siRNAs to GBM. Our results show that CTX-coupled SNALPs are more effective (both in vitro and in vivo) in mediating nucleic acid delivery to tumor cells than their nontargeted (NT) counterpart. Moreover, we demonstrate that SNALP-mediated miR-21 silencing in GBM/glioma cells increases the expression of the tumor suppressors PTEN and PDCD4, enhances caspase 3/7 activity, and, importantly, enhances the cytotoxic effect of the antiangiogenic drug sunitinib.ResultsPreparation and physicochemical characterization of targeted (CTX-coupled) and NT SNALPsWe have previously developed a lipid formulation composed of DODAP/DSPC/Chol/C16 mPEG2000 Ceramide (25:22:45:8, mol%), which was shown to efficiently encapsulate both siRNAs and asOs.15Mendonça LS Firmino F Moreira JN Pedroso de Lima MC Simões S Transferrin receptor-targeted liposomes encapsulating anti-BCR-ABL siRNA or asODN for chronic myeloid leukemia treatment.Bioconjug Chem. 2010; 21: 157-168Crossref PubMed Scopus (79) Google Scholar Here, locked nucleic acid (LNA)-modified asOs or siRNAs were encapsulated into this lipid formulation. Aiming at achieving specific tumor-targeting and increasing intracellular delivery, the ligand CTX was attached to the liposomal surface. In this regard, when liposomes, encapsulating anti-miR-21 oligonucleotides or siRNAs, were incubated with 4 mol% of micelles, values of 5.4 ± 2.8 and 5.0 ± 3.0 nmol CTX/µmol total lipid (respectively) and ~500 molecules of CTX per liposome were obtained. As shown in Table 1, the postinsertion step did not interfere with the loading of the encapsulated nucleic acids, since high encapsulation yields were obtained for anti-miR-21 oligonucleotides and siRNA in both targeted (89.3 ± 19.8 and 87.5 ± 11.1%, respectively) and NT (82.2 ± 15.8 and 85.5 ± 17.1%) formulations. Moreover, the SNALPs exhibited a net surface charge close to neutrality, with lower values of ζ potential for NT formulations (encapsulating siRNAs or anti-miR-21 oligonucleotides) as compared with CTX-coupled formulations (Table 1).Table 1Physicochemical characterization of CTX-coupled and NT liposomes encapsulating anti-miR-21 oligonucleotides (anti-miR-21) or anti-survivin siRNAsCTX-coupled SNALPsNT SNALPsSize (nm)PD indexEncapsulation efficiency (%)Zeta potential (mV)Size (nm)PD indexEncapsulation efficiency (%)Zeta potential (mV)Anti-miR-21178. 1 ± 21. 040.298 ± 0.106089. 30 ± 19. 783. 782 ± 1. 879163. 8 ± 24. 970. 219 ± 0. 126682. 17 ± 15. 818. 255 ± 2. 075siRNAs144. 4 ± 20. 620. 214 ± 0. 109587. 52 ± 11. 104. 122 ± 2. 118130. 4 ± 12. 560. 143 ± 0. 05285. 54 ± 17. 127. 652 ± 1. 779CTX, chlorotoxin; NT, nontargeted; PD index, polydispersity index; siRNAs, small interfering RNAs; SNALPs, stable nucleic acid lipid particles. Experiments were performed as described in Materials and Methods. Values are the mean ± SD of at least three independent experiments. Open table in a new tab The developed formulations also revealed capacity to protect the nucleic acid molecules from nuclease degradation, since in the absence of the detergent C12E8, the intercalation of the probe SYBR Safe with the encapsulated anti-miR-21 oligonucleotides/siRNAs was reduced by ~87 and 92%, respectively. Results obtained from photon correlation spectroscopy revealed that all formulations exhibited a size under 180 nm, nanoparticles encapsulating siRNAs being generally smaller than those encapsulating anti-miR-21 oligonucleotides with a narrow distribution (polydispersity index <0.3) (Table 1). The insertion of protein conjugates onto the liposome surface resulted in a small increase of liposomal size: NT liposomes encapsulating anti-miR-21 oligonucleotides or siRNAs exhibited 163.8 ± 25.0 and 130.4 ± 12.6 nm, respectively, whereas CTX-coupled liposomes encapsulating anti-miR-21 oligonucleotides or siRNAs exhibited 178.1 ± 21.0 and 144.4 ± 20.6 nm, respectively. Nevertheless, particle aggregation (and consequent increase in size) was observed for CTX-coupled SNALPs 3 months after their preparation, whereas particle aggregation was observed to a lesser extent in the NT formulations (data not shown), thus indicating that the presence of CTX may decrease the stability of the formulation over time.Evaluation of cellular association of SNALPs by flow cytometryExtensive association was observed 4 hours after exposure of U87 cells (at 37 °C) to CTX-coupled liposomes encapsulating 0.5 µmol/l of oligonucleotides, which was further enhanced when cells were exposed to 1 µmol/l of oligonucleotides encapsulated in targeted SNALPs (~75% fluorescent cells, 9.6 ± 4.2-fold increase in fluorescence intensity) when compared with that observed in cells exposed to 1 µmol/l of oligonucleotides encapsulated in NT liposomes (~5% fluorescent cells, 1.6 ± 0.4) (Figure 1a,b), as assessed by flow cytometry. Similar results were obtained when GL261 mouse glioma cells were incubated with CTX-coupled or NT liposomes encapsulating 0.5 or 1 µmol/l of oligonucleotides (Figure 1c,d). In contrast, following cell incubation with SNALPs at 4 °C (Figure 1b) or with 1 µmol/l of oligonucleotides, either per se or encapsulated in liposomes associated with a smaller amount of CTX (1 mol% of micelles instead of 4 mol%) (data not shown), no significant cellular association was detected.To demonstrate that cellular association of CTX-coupled SNALPs was mediated by specific interaction with cellular receptors, U87 cells were preincubated with 20 µmol/l of free CTX to block the CTX receptors. A moderate decrease in cellular association (reflected in the decrease in fluorescence intensity) was observed when cells were exposed to free CTX before the addition of CTX-coupled liposomes encapsulating 1 µmol/l of oligonucleotides (7.4 ± 1.9) when compared with that detected in cells exposed to 1 µmol/l of targeted SNALPs (11.3 ± 3.0). Reduced extent of association was also observed in cells exposed to bovine serum albumin-coupled liposomes encapsulating 0.5 or 1 µmol/l of oligonucleotides (Figure 1e).Aiming at evaluating whether CTX-coupled SNALPs would specifically target tumor cells, experiments were performed to determine the extent of their association with the nonmalignant cell line HEK293T (human embryonic kidney). As demonstrated in Figure 1f,g, a significant decrease in the extent of cellular association was observed following incubation with CTX-coupled SNALPs when compared with that determined in U87 GBM cells exposed to similar amounts of targeted SNALP-formulated oligonucleotides. Similar results were obtained from parallel experiments performed with primary cultures of mouse astrocytes (Supplementary Figure S1).Evaluation of cellular internalization by confocal microscopyIn order to confirm the results obtained on targeting specificity of CTX-coupled SNALPs by flow cytometry, cell internalization studies were performed using confocal microscopy. The results shown in Figure 2 reveal that following incubation of U87 cells, at 37 °C, with rhodamine-labeled CTX-coupled liposomes encapsulating FAM-labeled anti-miR-21 oligonucleotides, intensive red (lipid) and moderate green (oligonucleotide) fluorescence was detected throughout the cell cytoplasm (Figure 2b,d), whereas residual fluorescence was detected in the cytoplasm of cells exposed to NT liposomes (Figure 2a,c). A similar pattern of internalization was observed in GL261 mouse and F98 rat glioma cells exposed to liposomes encapsulating FAM-labeled oligonucleotides (Supplementary Figure S2), while only residual fluorescence was detected in mouse primary astrocytes (Figure 2e,f) or HEK293T cells (Figure 2g,h) incubated under the same conditions. Moreover, reduced internalization was observed upon cell incubation with CTX-coupled liposomes at 4 °C (Figure 3c,f) or presaturation of the CTX receptor with excess of free CTX (20 µmol/l) (Figure 3b,e) when compared with that observed in cells exposed to CTX-coupled liposomes at 37 °C (Figure 3a,d).Figure 2Stable nucleic acid lipid particle (SNALP) internalization in U87 glioblastoma cells, HEK293T human embryonic kidney cells and mouse primary astrocytes. Cells were incubated with chlorotoxin (CTX)-coupled or nontargeted (NT) liposomes encapsulating FAM-labeled anti-miR-21 oligonucleotides (for 4 hours at 37 °C), rinsed twice with phosphate-buffered saline, stained with DNA-specific Hoechst 33342 (blue) and then observed by confocal microscopy. The panel shows representative images at ×40 magnification of (a–d) U87 cells incubated with either (a,c) rhodamine-labeled NT or (b,d) CTX-coupled liposomes at a final oligonucleotide concentration of (a,b) 0.5 or (c,d) 1 µmol/l. The yellow dots are most likely due to the cellular colocalization of lipid and nucleic acid. Representative images of (e,f) mouse astrocytes and (g,h) HEK293T cells incubated with 1 µmol/l of SNALP-formulated oligonucleotides. Results are representative of two independent experiments. Bars corresponds to 20 µm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Stable nucleic acid lipid particle (SNALP) internalization in U87 glioblastoma cells and effect of cell preincubation with free chlorotoxin (CTX). Cells were incubated with CTX-coupled liposomes encapsulating FAM-labeled anti-miR-21 oligonucleotides (for 4 hours), rinsed twice with phosphate-buffered saline, stained with DNA-specific Hoechst 33342 (blue) and then observed by confocal microscopy. The panel shows representative images at ×40 magnification of U87 cells exposed to targeted SNALP-formulated oligonucleotides at 37 °C (a,d) either per se (37 °C) or (b,e) following preincubation with 20 µmol/l of free CTX for 1 hour (37 °C + CTX). (c,f) Cells exposed to targeted SNALP-formulated oligonucleotides at 4 °C. Results are representative of two independent experiments. Bars corresponds to 20 µm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although aggregation has been observed for CTX-coupled SNALPs 3 months after their preparation, images obtained by confocal microscopy (Supplementary Figure S3) and data from quantitative PCR showing a decrease in miR-21 expression levels in cells exposed to these SNALPs (data not shown) suggest that this formulation was internalized by the cells.Cellular association and internalization studies of liposomes encapsulating siRNAs also revealed enhanced particle uptake in cells exposed to increased concentrations of siRNAs encapsulated in CTX-coupled liposomes when compared with their NT counterpart (Supplementary Figure S4).MiR-21 silencing mediated by CTX-coupled SNALPs and its effect on the expression of the target proteins PTEN and PDCD4Having shown that CTX-coupled SNALPs efficiently deliver oligonucleotides to GBM/glioma cells, we evaluated whether intracellularly delivered anti-miR-21 oligonucleotides could modulate the expression of mature miR-21. As illustrated in Figure 4a,b, incubation of U87 and GL261 cells with 0.25 µmol/l of SNALP-formulated anti-miR-21 oligonucleotides resulted in a significant decrease in miR-21 levels (0.17 ± 0.22 and 0.21 ± 0.09, respectively), which was further enhanced with increasing concentrations of anti-miR-21 oligonucleotides (0.5 and 1 µmol/l). Parallel experiments demonstrated that cell exposure to NT liposomes encapsulating anti-miR-21 oligonucleotides did not considerably affect the levels of miR-21 (Supplementary Figure S5).Figure 4MiR-21 and PTEN/PDCD4 expression in U87 glioblastoma and GL261 glioma cells following incubation with chlorotoxin (CTX)-coupled liposomes encapsulating anti-miR-21 oligonucleotides. (a,b) MiR-21 and (c) PTEN/PDCD4 mRNA expression levels in (a) U87 and (b) GL261 cells, 48 hours after cell incubation with CTX-coupled liposomes encapsulating anti-miR-21 or scrambled oligonucleotides (n = 3). MiR-21 expression levels, normalized to the reference snord44 (human) or snord110 (mouse), and PTEN and PDCD4 expression levels, normalized to the reference HPRT1, are presented as relative expression values to control untreated cells. (d) Representative gel showing PTEN and PDCD4 protein levels in U87 (upper panel) and GL261 (lower panel) cells 48 hours after cell incubation with CTX-coupled liposomes encapsulating anti-miR-21 or scrambled oligonucleotides (n = 3). (e) Quantification of PTEN and PDCD4 bands observed in d, corrected for individual α-tubulin signal intensity. Results are presented as PTEN and PDCD4 expression levels relative to control. Values are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 to cells incubated with a similar amount of CTX-coupled liposomes encapsulating scrambled oligonucleotides.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MiR-21 silencing was also reflected on the expression of two of its targets, the tumor suppressors PDCD4 and PTEN.20Lu Z Liu M Stribinskis V Klinge CM Ramos KS Colburn NH et al.MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene.Oncogene. 2008; 27: 4373-4379Crossref PubMed Scopus (620) Google Scholar,21Meng F Henson R Wehbe-Janek H Ghoshal K Jacob ST Patel T MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer.Gastroenterology. 2007; 133: 647-658Abstract Full Text Full Text PDF PubMed Scopus (2317) Google Scholar As shown in Figure 4c, a moderate increase in PTEN mRNA levels was observed in both U87 (~15%, P > 0.05) and GL261 (25%, P < 0.05) cells incubated with 1 µmol/l anti-miR-21 oligonucleotides as compared with those observed when cells were exposed to a similar amount of scrambled oligonucleotides. Although no significant changes were observed in U87 cells, a small increase in PDCD4 mRNA was obtained in GL261 cells under the same experimental conditions (~20%, P > 0.05). More importantly, a considerable and significant increase in PDCD4 protein expression was observed in both U87 (25%, P < 0.05) and GL261 (30%, P < 0.01) cells incubated with 1 µmol/l of anti-miR-21 oligonucleotides when compared with that observed in cells transfected with a scrambled sequence (Figure 4d,e).Similarly to what was observed with the intracellularly delivered anti-miR-21 oligonucleotides, CTX-coupled liposome-mediated anti-survivin-siRNA delivery resulted in decreased levels of survivin mRNA (Supplementary Figure S4).Evaluation of caspase activation and apoptosis in tumor cell lines with reduced miR-21 expressionSince miR-21 has been proposed to play an antiapoptotic role in GBM, we investigated whether miR-21 silencing mediated by CTX-coupled SNALPs would affect the activity of the effector caspases 3 and 7, crucial components of the apoptotic cell death. As shown in Figure 5a, incubation of U87 and GL261 cells with 1 µmol/l of SNALP-formulated anti-miR-21 oligonucleotides resulted in a twofold increase (~1.76 and 1.66, respectively) in caspase 3/7 activity (P > 0.05) as compared with that observed upon incubation to SNALP-formulated scrambled oligonucleotides. More importantly, silencing of miR-21 followed by cell exposure to 15 (U87) and 5 (GL261) µmol/l of the tyrosine kinase inhibitor sunitinib resulted in a considerable increase in caspase 3/7 activity (5.3 ± 2.3 and 4.8 ± 1.9, respectively) when compared with that observed for cells exposed to sunitinib, either per se (1.6 ± 0.7 and 2.1 ± 0.8) or in combination scrambled oligonucleotides (2.1 ± 1.1, P < 0.01 and 2.9 ± 0.8, P > 0.05).Figure 5Evaluation of caspase activation, apoptosis and tumor cell proliferation in U87 glioblastoma and GL261 glioma cells. Cells were incubated with chlorotoxin-coupled liposomes encapsulating anti-miR-21 or scrambled oligonucleotides for 4 hours, washed with phosphate-buffered saline (PBS) and further incubated for 24 hours with fresh medium. Cells were subsequently exposed to 15 (U87) or 5 µmol/l (GL261) of sunitinib for 24 hours, rinsed with PBS, after which caspase/cell death detection and cell viability assays were performed. (a) Caspase 3/7 activity in U87 and GL261 cells incubated with 1 µmol/l of stable nucleic acid lipid particle (SNALP)-formulated anti-miR-21 or scrambled oligonucleotides, either per se or in combination with sunitinib. Results, presented as relative fluorescence units with respect to control untreated cells, were normalized for the number of cells in each condition. (b) Cell death detection in U87 cells exposed to 1 µmol/l of SNALP-formulated anti-miR-21 or scrambled oligonucleotides. For each condition (control, scrambled/anti-miR-21 1 µmol/l), results are presented as percentage of viable, early/late apoptotic and necrotic cells. Representative cell death plots for U87 cells incubated with (c) SNALP-formulated scrambled and (d) anti-miR-21 oligonucleotides. The percentage of viable (lower left), early apoptotic (lower right), late apoptotic (upper right) and necrotic (upper left) cells in the cell population is indicated in the plots. (e) Cell viability, evaluated by the Alamar Blue assay (as described in Materials and Methods) immediately after cell incubation with sunitinib. Values are presented as means ± SD (n = 3). Scrambled/anti-miR-21 1 µmol/l + S15/5: cells transfected with scrambled or anti-miR-21 oligonucleotides and further incubated with 15 or 5 µmol/l sunitinib. **P < 0.01 compared to cells incubated with SNALP-formulated scrambled oligonucleotides and further treated with 15 µmol/l sunitinib.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, an increase in the percentage of late apoptotic (P > 0.05) and necrotic cells (P < 0.05) was observed in U87 cells incubated with 1 µmol/l of SNALP-formulated anti-miR-21 oligonucleotides (Figure 5b,d), compared to that observed for cells incubated to 1 µmol/l of SNALP-formulated scrambled oligonucleotides (Figure 5b,c). The presence of sunitinib, a fluorescently active molecule, interfered with the cytometric detection of FAM-labeled annexin V and propidium iodide and, therefore, no information could be obtained about apoptotic cell death in the presence of sunitinib.Evaluation of tumor cell death following miR-21 silencingWe further evaluated whether the increase in tumor suppressor expression and caspase activity observed following SNALP-mediated miR-21 silencing would correlate with changes in tumor cell proliferation. Initial experiments were performed by exposing GL261 cells to different concent" @default.
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• W2053287601 date "2013-01-01" @default.
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• W2053287601 title "Tumor-targeted Chlorotoxin-coupled Nanoparticles for Nucleic Acid Delivery to Glioblastoma Cells: A Promising System for Glioblastoma Treatment" @default.
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• W2053287601 doi "https://doi.org/10.1038/mtna.2013.30" @default.
• W2053287601 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3696908" @default.
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